Enalaprilat

Reagents were purchased from Sigma-Aldrich, St. Louis, MO. Solvents for liquid chromatography-mass spectrometry (LC-MS) analyses were of HPLC LC-MS grade. Enalaprilat (sc-205669) and (S)-Lisinopril-d5 (sc-220030) were purchased from Santacruz Biotechnology, Dallas, TX, United States.

HK-2 cells were stimulated with TMAO (300 µM), low glucose (5 mM, unstimulated) or high glucose (30 mM) for 24 h at 37 °C in 5% CO₂. The HK-2 cells were also treated with TMAO (300 µM) for 12 h followed by treatment with DMSO (vehicle), candesartan (10 µM, Santa Cruz Biotechnology), dapagliflozin (10 µM, Santa Cruz Biotechnology), losartan (10 µM, Santa Cruz Biotechnology) and enalaprilat (5 µM, Santa Cruz Biotechnology Inc) for an additional 12 h in the presence of TMAO. After 24 h, the cells were detached with Accutase (Sigma-Aldrich) and megalin was detected using the monoclonal mouse anti-human megalin/LRP2 Alexa Fluor 488-conjugated antibody (R&D Systems, Minneapolis, USA). Cells were stained with 0.25 µg antibody for 20 min at room temperature. The expression was evaluated using the Gallios (Beckman Coulter, Brea, CA, USA) flow cytometer with 488 nm laser and FL1 525/40 nm band-pass filter. The data were analyzed with Kaluza Flow Cytometry Analysis v1.3 (Beckman Coulter).